Aquatic birnavirus induces apoptosis through activated caspase-8 and -3 in a zebrafish cell line

In this study, the possible influence of temperature on infectious pancreatic necrosis virus (IPNV)-induced apoptosis in a zebrafish liver epithelium (ZLE) cell line was investigated. At a lower temperature (18 degrees C), there was expression of viral proteins VP2 and VP3 at 4 h post-infection (p.i.). At this time no expression was found in the high temperature group at 28 degrees C. The cell survival ratio was 52 and 18% at 24 and 48 h p.i., respectively, during IPNV infection at 18 degrees C. In addition, we assayed for apoptosis in IPNV-infected cells with terminal deoxynucleotidyl transferase (TdT)-mediated end labelling (TUNEL) of DNA at different dosages of virus. We found a ratio of apoptotic cells of 8 and 25% at 12 and 18 h p.i., respectively, in the multiplicity of infection (MOI) 1 group. The MOI 10 group had 20 and 45% apoptotic cells at 12 and 18 h, respectively. Furthermore, at 18 degrees C IPNV activated the caspase-8 and 3 from 1.5 to 2 times at 12 and 18 h p.i., respectively. Taken together, these findings suggest that successful virus replication occurs at the low temperature (18 degrees C) compared with the non-permissive temperature of 28 degrees C. Thus, IPNV replication is capable of activating caspase-8 and -3 and inducing host apoptosis.     

The persistence of infectivity of Tetracapsulabryosalmonae-infected water for rainbow trout, Oncorhynchus mykiss (Walbaum)

Proliferative kidney disease (PKD) is caused by the infection of susceptible salmonid fish with spores of the myxozoan Tetracapsula bryosalmonae , a parasite harboured and released by several species of bryozoans. Under natural conditions, PKD is a water-borne infection of fish, whose outcome and spatio-temporal dissemination depend on the viability of spores present in the water. In order to evaluate the duration of parasite infectivity, juvenile rainbow trout, Oncorhynchus mykiss , were exposed for 20 h to T. bryosalmonae -infected water at various times post-water collection or after different filtration procedures. When infected water was held in a temperature range of 14.5–17 °C for up to 14 days, PKD was transmitted to the fish only between 0 and 12 h post-water collection and its infectivity vanished between 12 and 24 h. Similarly, the infectivity of water passed through 25 μm but not through 1 μm mesh filters, and was lost in the material eluted from the 1 μm filtration membrane although the parasite's DNA was amplified from this material. The parasitic infectivity in water appears to be fragile and this may offer opportunities to decrease the impact of PKD in trout farms by the implementation of management procedures aimed at reducing the number of the bryozoan-holding surfaces located in the river, immediately upstream from these farms.     

Amelioration of LPS‐induced inflammatory response and oxidative stress by astaxanthin in Channa argus lymphocyte via activating glucocorticoid receptor signalling pathways

The present study was conducted to evaluate the effects of astaxanthin (AST) against lipopolysaccharide (LPS)‐induced lymphocyte viability, ultrastructural lesions, apoptosis, oxidative stress and inflammatory responses in Channa argus. Lymphocytes exposed to more than 10 μg/ml LPS alone for 24 hr showed significantly decreased cell viability, elevated nitric oxide (NO) and malondialdehyde (MDA), lactate dehydrogenase (LDH) contents, and increased nuclear factor κB p65 (NF‐κB p65), myeloid differential protein‐88 (MyD88), tumour necrosis factor‐α (TNF‐α), interleukin‐1β (IL‐1β), interleukin‐8 (IL‐8), caspase‐3, caspase‐8 and caspase‐9 gene expression. LPS at a concentration of 10 μg/ml could induce oxidative stress and inflammatory responses in lymphocytes. The activities of antioxidant enzymes (catalase (CAT), glutathione peroxidase (GPx), superoxide dismutase (SOD)) were significantly decreased after exposure to 10 μg/ml LPS. Besides, AST strikingly antagonized the LPS‐induced negative effects. AST significantly increased the expression of HSP70, HSP90, IκB‐α, and glucocorticoid receptor (GR) and decreased inflammatory responses. Further study showed that AST can activate GR signalling pathway and inhibit p65 phosphorylation. In addition, AST attenuated LPS‐induced apoptosis, mitochondrial swelling, degeneration and vacuolization. Collectively, these findings suggest that AST has protective roles in LPS‐induced cell damage via modulating GR activation in C. argus lymphocytes.     

Replication of infectious pancreatic necrosis virus in trout leucocytes and detection of the carrier state

Abstract. The exact cellular site of replication of infectious pancreatic necrosis virus (IPNV) in carrier fish is unknown. In order to determine if IPNV replicates in trout leucocytes, we purified leucocytes from normal (non-carrier) trout and separated the cells into an adherent and a non-adherent population. IPNV replicated in less than 0-01 % of the adherent leucocytes with a yield of about 400 p.f.u./cell. IPNV also became associated with less than 0.07% of the non-adherent leucocytes; either IPNV did not replicate in these cells or the yield was, at best, only a few p.f.u./cell. Trout persistently infected with IPNV (carrier fish) were tested for the presence of IPNV in leucocytes by co-cultivating with a sensitive fish cell line; this same population of trout was also tested for IPNV by organ sampling using standard methods. Ninety-eight per cent of the trout were positive for IPNV by organ sampling, but only 75 % yielded IPNV from leucocytes. Thus a blood sample from a living fish can be used to detect the presence of IPNV.     

Experimental study of the susceptibility of Atlantic cod, Gadus morhua (L.), to infection with an IPNV strain pathogenic for Atlantic salmon, Salmo salar L

Intraperitoneal (IP) injection, cohabitation and immersion routes of infection were used to determine if Atlantic cod, Gadus morhua (L.), of 1 and 3 g are susceptible to infectious pancreatic necrosis (IPN). Mortalities of cod injected IP were significantly higher when challenged with infectious pancreatic necrosis virus (IPNV) than with phosphate buffered saline. This is the first report of Atlantic cod mortalities caused by IPNV. Fish challenged by cohabitation had significantly higher mortalities than the controls, but mortalities of Atlantic cod challenged with IPNV by immersion were not significantly different from controls. Titres of IPNV in the tissues of infected fish were sometimes very high (range 10²–10¹⁰ infectious units per gram of tissue) suggesting virus replication and titres of fish that died were generally higher than those of fish which survived. However, the relatively low mortality rates when challenged by cohabitation and immersion (20% and 17%, respectively), compared to the IP injection challenge (100%) suggest that 1 and 3 g cod have low susceptibility to IPN when challenged by more natural routes. These data strongly suggest that the cause of death of experimentally challenged cod was IPNV and further histological evidence for this came from 1 g cod challenged IP with IPNV in which the pancreas showed severe necrosis and heavy immunostaining for IPNV coincidentally with the peak of mortalities.     

Salmon cells SHK‐1 internalize infectious pancreatic necrosis virus by macropinocytosis

We have previously shown that infectious pancreatic necrosis virus (IPNV) enters the embryo cell line CHSE‐214 by macropinocytosis. In this study, we have extended our investigation into SHK‐1 cells, a macrophage‐like cell line derived from the head kidney of Atlantic salmon, the most economically important host of IPNV. We show that IPNV infection stimulated fluid uptake in SHK‐1 cells above the constitutive macropinocytosis level. In addition, upon infection of SHK‐1 cells, IPNV produced several changes in actin dynamics, such as protrusions and ruffles, which are important features of macropinocytosis. We also observed that the Na+/H+ pump inhibitor EIPA blocked IPNV infection. On the other hand, IPNV entry was independent of clathrin, a possibility that could not be ruled out in CHSE 214 cells. In order to determine the possible role of accessory factors on the macropinocytic process, we tested several inhibitors that affect components of transduction pathways. While pharmacological intervention of PKI3, PAK‐1 and Rac1 did not affect IPNV infection, inhibition of Ras and Rho GTPases as well as Cdc42 resulted in a partial decrease in IPNV infection. Further studies will be required to determine the signalling pathway involved in the macropinocytosis‐mediated entry of IPNV into its target cells.     

Use of cloned cDNA probes for diagnosis of infectious pancreatic necrosis virus infections

Abstract. A dot-blot hybridization test has been developed for the detection of infectious pancreatic necrosis virus (IPNV) in infected fish. For this purpose, cloning of the dsRNA of the West Buxton strain of IPNV was carried out. Two cDNA clones (WB and A4) were characterized for use as diagnostic probes and corresponded to IPNV genome segments A and B. respectively. Clone WB1, with an insert of 812 base pairs, showed an 87 and 77% nuclcotidc sequence homology with the corresponding sequences of Jasper and N1 strains, respectively. Clone A4, with an insert size of 596bp, presented a nuclcotidc sequence homology of 90 and 80% with the corresponding sequences of the Jasper and Sp strains, respectively. Both probes were able to detect 15 ng of purified dsRNA, and were highly efficient in detecting the RNA of American IPNV strains. However, the A4 probe was less effective than WB1 in hybridizing to RNA from European and Spanish strains of IPNV. Both probes detected IPNV RNA in cells 4–8h post-infection with the homologous West Buxton strain, 8–12h post-infection with other American strains and 24h post-infection with the European strains of IPNV. The method was less sensitive in detecting IPNV RNA directly in infected fish tissues. However, the present authors obtained a 100% effectiveness to detect viral RNA in cells inoculated with fish tissues confirmed by conventional diagnostic methods as being infected with IPNV. Therefore, the hybridization test is appropriate if combined with conventional diagnostic procedures, e.g. applying the dot blot hybridization test on tissue cultures 12–24 h after inoculation with infected fish tissue homogenates.     

Effect of hyperoxygenation and low water flow on the primary stress response and susceptibility of Atlantic salmon Salmo salar L. to experimental challenge with IPN virus

Intensive salmon smolt production normally includes reduced water flow and hyperoxygenation (added oxygen) of remaining water. There is little information on how different water quality parameters influence the fish health and the susceptibility to infectious diseases. The current experiment was carried out to evaluate if the combination of hyperoxygenation and reduced water flow (hyperoxic) can act as a chronic stressor to salmon in freshwater (FW) in such a way that it increases the susceptibility to IPN virus (IPNV) following seawater transfer. In FW, after 22 days of hyperoxic exposure plasma ion, TBARS and cortisol were measured. The cortisol levels were significantly (p = 0.011) higher in the hyperoxic group compared to controls maintained under normal oxygen saturation and water flow (normoxic), indicating chronic stress. Hyperoxygenation in FW caused decreased plasma [Cl⁻] compared to the normoxic group (p = 0.037), while [K⁺] tended to be higher in the hyperoxic group (p = 0.088). No significant differences were observed in plasma [Na⁺], total osmolality, TBARS or hematocrit, but there was a tendency towards a lower hct in the hyperoxic compared to the normoxic group. In SW the mortality was higher in the hyperoxic group challenged with IPNV (34%) compared to the normoxic group challenged with IPNV (20%) (p = 0.02), and no mortality was observed in the PBS injected fish. The challenged fish showed an overall increase in plasma cortisol day 8, 10, 12 and 14 post-challenge (p = 0.015, p = 0.000, p = 0.046 and p = 0.022 respectively). After SW transfer and challenge, plasma [K⁺] was elevated in both challenged groups, but no consistent trends were found for plasma [Cl⁻], [Na⁺] or total osmolality during the SW phase. There were no significant differences in the gene expression level of IFN 1, Mx and IL 1β prior to challenge, suggesting that the basic expression level of these genes were not affected by hyperoxygenation. IPNV was detected in kidney and pylorus, by immunohistochemistry, cell culture, and RT-PCR in head kidney. This experiment indicates that chronic stress induced by a combination of low water flow and hyperoxygenation increases the susceptibility to IPNV challenge.     

Quantitative analysis of an experimental white spot syndrome virus (WSSV) infection in Penaeus monodon Fabricius using competitive polymerase chain reaction

White spot syndrome virus (WSSV) has been a major pathogen of cultured Penaeus monodon Fabricius in Malaysia since 1994. As quantitative study on the replication of WSSV is in its infancy, competitive polymerase chain reaction (PCR) was used for quantitative study of an experimental WSSV infection per os in growout P. monodon . Gills, abdominal integument and abdominal muscle were selected for viral quantification. Infection was detectable as early as 14 h postinfection (h p.i.) in both gills and integument, but the infection in muscle was only detected at 24 h p.i. Gill tissue had the highest viral load, followed by integument and muscle. Typical viral growth curves were obtained for all organs with distinct phases of eclipse (0–24 h p.i.), logarithmic (24–48 h p.i.) and the plateau (48–120 h p.i.). Cumulative mortality rapidly increased from 48 h p.i. and reached 100% at the end of the plateau phase at 120 h p.i. Gross signs of white spots and reddish discoloration were also obvious in moribund individuals from the plateau phase. Based on the three phases of viral growth, WSSV infection was classified into light, moderate and heavy infection stages.     

Occurrence and prevalence of infectious pancreatic necrosis virus in rainbow trout (Oncorhynchus mykiss) cultured in cages in the Black Sea

Rainbow trout (Oncorhynchus mykiss) cultured in cage systems in the South Eastern Black Sea were surveyed for the type, occurrence and prevalence of infectious pancreatic necrosis virus (IPNV). Two nearby farms (designated as Farm A and Farm B) were visited monthly in 2007 and 2008. At each farm, 385 fish were selected randomly from five cages. Another farm with infected trout from a hatchery also was monitored for IPNV from the transfer to harvest. IPNV was found to be prevalent in both farms surveyed. In Farm A, IPNV was present throughout the growing period, from January to May, and all five randomly sampled cages tested positive for IPNV in March and April of 2007. In Farm B, IPNV was present only in February and March in 2007, and in 2008, IPNV was observed in January (two cages) and February (one cages) at low levels. Interestingly, IPNV was absent 2 weeks after transfer to the sea at 17.5°C. The same strain of IPNV, genotype III that was isolated from the same stock of fish at the hatchery, reoccurred when water temperatures dropped to 12°C in December in the Black Sea. Transferring fish to the sea at high water temperatures could lessen the negative impacts of IPNV on growth of rainbow trout in brackish water.     

鲤Rhbdd3蛋白的抗病毒功能

Rhbdd3(Rhomboid domain-containing protein 3)蛋白在哺乳动物天然免疫中发挥了重要作用,但水生动物中rhbdd3基因的确定序列及Rhbdd3蛋白的功能均尚未见报道。为研究鲤(Cyprinus carpio)的Rhbdd3蛋白在鱼类细胞中的功能,探讨其过表达对鱼类病毒感染的影响,本研究通过PCR扩增得到了鲤rhbdd3基因的编码序列,并将其克隆至pCI-neo载体上,构建了真核表达质粒pCI-rhbdd3。pCI-rhbdd3转染鲤上皮瘤细胞EPC(epithelioma papulosum cyprinid)和鲑囊胚细胞CHSE-214(chinook salmon embryo)后利用制备的特异性抗体进行Western blot,检测Rhbdd3蛋白的表达情况,并利用CCK-8试剂检测其过表达对细胞增殖的影响。转染后分别进行鲤春病毒血症病毒(SVCV)和传染性胰腺坏死病毒(IPNV)的感染实验,并利用间接免疫荧光、Western blot和RT-qPCR方法检测Rhbdd3过表达对SVCV和IPNV增殖的影响。结果显示,pCI-rhbdd3转染后Rhbdd3蛋白在EPC和CHSE-214细胞中得到了过表达,且Rhbdd3蛋白的过表达能显著抑制SVCV和IPNV的复制,但不影响两种细胞的正常活性。本研究为鱼类广谱抗病毒药物的开发提供了新的实验依据,也为鱼类抗病毒新品种的培育奠定了重要基础。     

Identification and pathogenicity of Vibrio parahaemolyticus isolates and immune responses of Penaeus (Litopenaeus) vannamei (Boone)

Five different Vibrio parahaemolyticus strains (SH8, SH108, SH58, AH5 and GD10) isolated from the hepatopancreas of moribund shrimp in farms of mainland China were identified and capable of inducing massive mortality of Penaeus (Litopenaeus) vannamei. The immersion challenge results with five isolates indicated variance of virulence, while only GD10 caused massive sloughing of tubule epithelial cells which was recognized as the most significant symptom of AHPND. Differences in immune responses were detected of P. vannamei during 48 h post‐infection (p.i.) by injection or immersion challenge with V. parahaemolyticus (SH8, SH108 and GD10) isolates. When injected SH8 and SH108 isolates, the expression of lysozyme (LSZ) showing statistically significant upregulation at 16 and 48 h p.i. and that of Toll‐like receptors (TLR) showed statistically significant upregulation at 48 h p.i. When immersion challenge with the GD10 isolate, TLR were upregulated after 8 h p.i. challenge with 10⁴ cfu mL^?1; however, LSZ was downregulated when challenged with 10³ cfu mL^?1. The results suggested that LSZ and TLR serve as crucial molecular markers of innate immunity in shrimp against V. parahaemolyticus infection. LSZ is a vital marker for acute bacterial infection, while TLR serves as a crucial marker for chronic infection.     

Design and validation of a RT‐qPCR procedure for diagnosis and quantification of most types of infectious pancreatic necrosis virus using a single pair of degenerated primers

Infectious pancreatic necrosis virus (IPNV) is an important virus which affects the salmonid aquaculture industry worldwide; therefore, it is important to develop rapid and reliable methods of diagnosis to detect the disease at early stages. Nowadays, RT‐qPCR is replacing other methods because it provides additional information on the viral load, which is important to have a better understanding of the virus replication level and of the stage of the infection and its risk level. The main problem stems from the high diversity of this virus, which can compromise the reliability of the diagnosis. In this study, we have designed an RT‐qPCR procedure for diagnosis and quantification of IPNV based on a single pair of primers targeted to segment B. The procedure has been validated, in vitro and in vivo, testing two different types of standards against seven reference strains and 23 field isolates from different types. The procedure is reliable for the detection of any type, with a detection limit of 31 TCID₅₀ mL^?1, 50 pfu mL^?1 or 66 RNA copies mL^?1, depending on the standard. All the standard curves showed high reliability (R² > 0.95). The results support the high reliability of this new procedure for the diagnosis and quantification of IPNV.     

Tetrahymena sp. infection in guppies, Poecilia reticulata Peters: parasite characterization and pathology of infected fish

Tetrahymena sp. infection was diagnosed in guppies imported from Singapore. The parasite was isolated (Tet-NI) and optimally cultured in vitro in RM-9 medium. Cytological analyses [silver-staining and scanning electron microscopy (SEM)] revealed a pyriform-shaped, 64 × 41-μm holotrich ciliate without caudal cilium, containing a macro-nucleus (18.25 × 16.83 μm) and micro-nucleus (5.73 × 5.40 μm). Wet-mount examination and histological analyses of fish exposed to the parasite by co-habitation, immersion and infection by i.p. (intra-peritoneal) and i.m. (intra-muscular) injection revealed numerous ciliates on the skin, and in the gill and caudal fin blood vessels. Ciliates surrounded internal organs, the peri-orbital region of the eye, and were observed inside developing guppy embryos. Some muscle necrosis was associated with infection, but little or no inflammatory response. Immersion, co-habitation and i.m. injection caused relatively high infection rates and levels in the skin and tail, and lower infection in the gill blood vessels and internal organs; i.p. injection caused higher infection in the gill blood vessels and internal organs. Co-habited fish had relatively high infection levels in the hind-gut sub-mucosa. This is the first report of controlled systemic infection by Tetrahymena sp.     

Observations on the effect of salinity and photon fluence rate on the induction of sporulation and rhizoid formation in the green alga Enteromorpha prolifera (Müller) J. Agardh (Chlorophyta, Ulvales)

ABSTRACT: Although most members of the genus Enteromorpha are important edible green algae, some species are also potentially economically valuable crops. Samples of E. prolifera were obtained from the Yoshino River estuary, Tokushima, Japan and cultured in laboratory conditions at 10°C, a salinity of 20 psu, under white light with 12 h light : 12 h dark cycle and at a photon fluence rate (PFR) of 40 μmol/m² per s for 30 days. In the present study, the effect of salinity and PFR on the induction of reproductive cells and rhizoid formation were investigated. Synchronous formation of swarmers by thalli was induced in excised disks of 1.2 mm diameter after 2–5 days incubation. The optimum salinity for maturation of reproductive cells was between 5.0 and 52.0 psu, and between 13.2 and 45.3 psu for swarmer release, although the lower limit for swarmer release was 5.0 psu. Maturation of reproductive cells and swarmer release required a PFR higher than 16 μmol/m² per s. The minimum PFR for swarmer release was 8 μmol/m² per s. Many rhizoids were formed between 1.6 and 52.0 psu and photon fluence rates between 8 and 320 μmol/m² per s. Rhizoids were formed in a polarized manner.     

Short‐term Effects of Genistein on the Reproductive Characteristics of Male Gibel Carp,Carassius auratus gibelio

The objective of this study was to determine the effects of genistein or 17β‐estradiol (E2) on the reproductive physiology in male gibel carp, Carassius auratus gibelio. Maturing male gibel carp received intraperitoneal injections of E2 (10 µg/g body weight), one of two genistein doses (5 µg/g body weight, G5, or 50 µg/g body weight, G50), or the injection vehicle every other day for 10 d. Disruptions in reproductive capacity were determined by measuring indices of sperm quality, plasma metabolites and sex steroids, histological analyses of testes, fertilization rate, and offspring viability. E2 and genistein treatment reduced gonadosomatic index and milt volume, while reduction in spermatozoa concentration and spermatocrit occurred only in E2 and G50‐treated males. Histological examination of the testes indicates that E2 and genistein inhibited reproductive capacity through disruption of the spermatogenesis in males. Genistein reduced fertilization rate and offspring viability at 6 d after hatch (d.a.h.). Plasma testosterone and E2 decreased and increased, respectively, with E2 and G50 treatment. E2 and G50 treatment altered plasma metabolite phosphorus, calcium, cholesterol, and triglyceride. These findings indicate that genistein can negatively affect reproductive capacity in male gibel carp, suggesting that high dietary genistein may impair gonad development.     

Vaccination of rainbow trout, Oncorhynchus mykiss (Walbaum), with recombinant and DNA vaccines produced to Flavobacterium psychrophilum heat shock proteins 60 and 70

Flavobacterium psychrophilum heat shock proteins (Hsp) 60 and 70 are highly immunogenic and were therefore investigated as potential vaccine candidates. Recombinant Hsps were purified from Escherichia coli and rainbow trout ( Oncorhynchus mykiss ) were intraperitoneally injected with phosphate buffered saline/Freunds complete adjuvant (FCA), 8 μg of rHsp60/FCA, rHsp70/FCA or a combination of 4 μg each of rHsp60 and rHsp70/FCA. Antibody responses against recombinant Hsp60 and Hsp70 8 weeks post-immunization were observed, but only fish immunized with rHsp70 exhibited highly elevated antibody levels against F. psychrophilum whole cell lysate. Some cross reactivity occurred, which may have been due to the V5 tag common to both proteins. Protection against F. psychrophilum challenge was not observed in any treatments at 8 weeks post-immunization. To further investigate any protective effect of these proteins, hsps were polymerase chain reaction amplified and cloned into pVAX1. Rainbow trout were intramuscularly injected with 8 μg of pVAX1hsp60, pVAX1hsp70 or a combination of 4 μg each of pVAX1hsp60 and pVAX1hsp70. Antibody responses at 4 weeks post-immunization were low and protection was not observed following challenge at 6 or 10 weeks post-immunization. Although Hsps of F. psychrophilum have been shown to be immunodominant, these antigens do not appear to be good vaccine candidates when delivered alone or in combination.     

Continuous exposure to infectious pancreatic necrosis virus during early life stages of rainbow trout, Oncorhynchus mykiss (Walbaum)

Rainbow trout, Oncorhynchus mykiss (Walbaum), were exposed continuously to infectious pancreatic necrosis virus (IPNV) at 0, 10¹, 10³ or 10⁵ plaque forming units (pfu) L⁻¹ of water to estimate the effects of chronic IPNV exposure on early life stages. Fish density averaged 35 fish L⁻¹ (low density) or 140 fish L⁻¹ (high density), and the tank flow rate was 250 mL⁻¹ min. Virus exposure began at 6 days before hatch and continued until fish were 44 days old. Cumulative per cent mortality, analysis of survival and hazard functions, and discrete-time event analysis were used to explore the patterns of survival and mortality. In eggs and fish exposed to IPNV, mortality significantly greater than in the 0 pfu L⁻¹ exposure did not occur until IPNV concentration was 10⁵ pfu L⁻¹ at low fish density and 10³ pfu IPNV L⁻¹ at high fish density. These results suggest that in the natural aquatic environment, where rainbow trout densities are likely to be considerably lower than in this study, mortality resulting from infection with IPNV will very likely not occur when ambient concentrations of virus are ≤10³ pfu IPNV L⁻¹. In aquaculture rearing units, trout density is likely to be as high or higher than the densities used in this study. Therefore, continuous inputs of virus at concentrations greater than 10¹ pfu L⁻¹ may result in IPN epidemics in aquaculture facilities.     

Histology,immunocytochemistry and qRT‐PCR analysis of Atlantic salmon,Salmo salar L., post‐smolts following infection with infectious pancreatic necrosis virus (IPNV)

Infectious pancreatic necrosis (IPN) is a very serious viral disease in terms of its impact on production of Atlantic salmon, Salmo salar L., fry and post‐smolts. Post‐smolts of Atlantic salmon were injected with infectious pancreatic necrosis virus (IPNV) and cohabited with naive fish to produce natural infection. Cohabitant fish were sampled every 2 days, up to day 36 post‐infection (p.i.). From 90 cohabitant fish, 11 (12.2%) were positive by immunohistochemistry (IHC). The first detection of IPNV by IHC occurred on day 16 p.i. which coincided with the onset of mortality in this group. Besides the pancreas, the liver was found to be a key target organ for IPNV. For the first time, the virus was observed in the islets of Langerhans and in the kidney corpuscles of Stannius which suggests that the virus could affect the fish’s metabolism. The liver of two fish, which showed the most widespread presence of IPNV by IHC, had a pathology including focal necrosis and widespread presence of apoptotic hepatocytes, many of which did not stain for virus by IHC. Up‐regulation of cytokine gene expression was found only in the IHC‐positive (IHC+ve) fish and reflected the level of infection as determined by IHC positivity of the liver. In most fish, interferon (IFN), Mx, γIFN and γIP were up‐regulated in liver and kidney, while only IFN and Mx were up‐regulated in gill. IL1β and TNFα were not induced in any tissue. The gill showed variable levels of constitutive expression of IL1β and γIFN. The two fish with liver pathology had the highest level of IFN expression, especially relative to the level of Mx expression, in the liver compared with the other IHC+ve fish which did not have a liver pathology. The results suggest that following widespread infection of hepatocytes, the cells may over‐produce IFN, resulting in apoptosis of neighbouring cells with subsequent death from liver failure.